Mitochondria are the main sources of energy in the cell. They contain their own DNA (mtDNA), whose genes encode components of the respiratory chain/oxidative phosphorylation system. They are essential for the normal functioning of all cells in the body, and are absolutely critical for the function of those tissues that are highly dependent on aerobic metabolism, especially muscle and brain. Since 1988, both mtDNA point mutations and mtDNA rearrangements (i.e. large-scale deletions and duplications) have been associated with a heterogeneous group of sporadic, mendelian, and maternally-inherited mitochondrial encephalomyopathies. These mutations generally cause an impairment of the respiratory chain, with a reduction in ATP synthesis. However, very little is known about how affected cells cope with the reduced ATP production: for example, which ATP-dependent cellular functions are preserved and which are down-regulated or abolished. Understanding ATP distribution inside mutant, as well as normal, cells would be extremely important for the interpretation of the biochemical and clinical phenotype of mitochondrial disorders. This Career Development Award Application proposes to investigate the effect of mtDNA abnormalities, on the intracellular ATP pool in different cell compartments, with particular emphasis on the mitochondria, the cytoplasmic membrane, and the nucleus. We plan to study the ATP content in cytoplasmic hybrids of human mtDNA- less cyss ( rho o cells ) repopulated with mitochondria derived from patients tissues, by targeting a recombinant firefly luciferase to different cell compartments. Utilizing a similar experimental approach, we will also attempt a novel genetic strategy for treatment of point mutations in the mtDNA ATPase6 gene, that are responsible for a maternally- inherited form of Leigh syndrome (MILS): to recode the ATPase 6 gene to contain the universal genetic code by in vitro mutagenesis, to fuse a mitochondrial targeting sequence to the recoded sequence, and then to transfer this construct into the nucleus, in order to express the gene from nuclear DNA and target it back to mitochondrial ( allotopic expression). Allotopic expression of the recoded wild-type genes should partially restore the APT synthetic function in mutant cells.